1. Field of the Invention
The present invention relates to a two-photon probe for real-time monitoring of intracellular magnesium ions, a method for preparing the two-photon probe, and a method for real-time monitoring of intracellular magnesium ions using the two-photon probe. More specifically, the present invention relates to a two-photon probe that has a sufficiently low molecular weight to stain cells and is suitable for real-time imaging of intracellular magnesium ions due to its very high two-photon fluorescence efficiency, a method for preparing the two-photon probe, and a method for real-time monitoring of intracellular magnesium ions using the two-photon probe.
2. Description of the Related Art
Mg2+ is one of the most abundant divalent metal ions in cells, and it plays crucial roles in many cellular processes such as proliferation and cell death as well as participating in the regulation of hundreds of enzymatic reactions. To detect intracellular Mg2+, a variety of membrane-permeable fluorescent probes have been developed with some of them being commercially available (The Handbooks—A Guide to Fluorescent Probes and Labeling Technologies, 10th ed.; Haugland, R. P. Ed.; Molecular Probes: Eugene, Oreg., 2005.; H. Komatsu, N. Iwasawa, D. Citterio, Y. Suzuki, T. Kubota, K. Tokuno, Y. Kitamura, K. Oka, K. Suzuki, J. Am. Chem. Soc. 2004, 126, 16353-16360.; G. Farruggia, S. Iotti, L. Prodi, M. Montalti, N. Zaccheroni, P. B. Savage, V. Trapani, P. Sale, F. I. Wolf, J. Am. Chem. Soc. 2006, 128, 344-350.). Most of them are used as their acetoxymethyl (AM) esters, which can readily undergo enzymatic hydrolysis to regenerate the metal-ion probe inside the cell. However, confocal microscopy with one-photon (OP) fluorescent probes is limited for use near the tissue surface (<100 μm).
To observe cellular events deep inside the tissue, it is crucial to use two-photon microscopy (TPM). TPM employing two near-infrared photons for excitation offers a number of advantages over one-photon microscopy, including increased penetration depth (>500 μm), lower tissue autofluorescence and self-absorption, as well as reduced photodamage and photobleaching (W. R. Zipfel, R. M. Williams, W. W. Webb, Nat. Biotechnol. 2003, 21, 1369-1377; F. Helmchen, W. Denk, Nat. Methods, 2005, 2, 932-940.).
The extra penetration that TPM affords is of particular interest in tissue imaging because surface preparation artifacts such as damaged cells extends over 70 μm into the brain slice interior (R. M. Williams, W. R. Zipfel, W. W. Webb, Curr. Opin. Chem. Biol. 2001, 5, 603-608.). However, most of the OP fluorescent probes presently used for TPM have small two-photon action cross sections (φδ) that limit their use in TPM. Another limitation associated with tissue imaging is a mistargeting problem, which results from membrane-bound probes (The Handbooks—A Guide to Fluorescent Probes and Labeling Technologies, 10th ed.; Haugland, R. P. Ed.; Molecular Probes: Eugene, Oreg., 2005.; J. R. Long, R. S. Drago, J. Chem. Ed. 1982, 59, 1037-1039; K. J. Hirose, Incl. Phenom. Macrocycl. Chem. 2001, 39, 193-209.). As the probes can be accumulated in any membrane-enclosed structure within the cell and as the fluorescence quantum yield should be higher in the membrane than in the cytosol, it is practically difficult for the signals from membrane-bound probes to be separated from those of the probe-Mg2+ complex.
Therefore, there is a need to develop efficient two-photon probes with 1) enhanced φδ values for brighter TPM images and 2) larger spectral shifts in different environments for better discrimination between the cytosolic and membrane-bound probes.